1. Field of the Invention
The invention relates to a method of analysing a liquid biological medium such as human blood in order to detect or identify viruses therein having antigen properties (i.e. virus antigens) or erythrocyte or cell antigens or antibodies.
In medical analysis, a frequent problem is the exact determination of the nature of the antigens carried by a cell or particle, and the nature of the antibodies or virus antigens in a serological medium. This applies inter alia to blood transfusions, where it is essential to ensure that the blood of the donor or donors is compatible with the blood of the future recipient.
Any incompatibility between the two blood systems is because the plasma of one blood system may contain molecules called "antibodies" which may become fixed on to complementary structural units or "antigen units" or "antigens" in the membranes of the red corpuscles or erythrocytes in the other blood system. An antigen of this kind is called an "erythrocyte antigen". An antibody capable of becoming fixed only on to a particular antigen is called an antibody "specific" to the antigen. Antibodies, if fixed on to antigen units carried by erythrocytes, may agglutinate them and destroy or seriously injure them, and may thus produce accidental illness during blood transfusions.
It is also essential to ensure that the donor's blood cannot transmit to the recipient certain diseases which are identified by detecting a virus antigen (i.e. a virus having antigenic properties) or an antibody specifically corresponding to the disease. One such disease is post-transfusion hepatitis, which may occur in the recipient if the donor's blood contains a virus called HB virus, which carries the HB.sub.s antigen.
This clearly shows the great importance of examining the blood of donors and recipients to detect any kinds of antibodies, cellular antigens or viruses having antigenic properties (i.e. virus antigens) which are capable of producing incompatibility reactions or disease in the recipients.
To make a transfusion, therefore, it is essential to be protected from any lack of sensitivity of the detection method. In addition, there should be practically no risk of error in determining the specificity of erythrocyte or cell antigens, antibodies or viruses (or virus antigens).
For the sake of clarity, we shall define certain terms used in the following description.
"Virus having antigenic properties" means a virus which reacts with a known specific antibody. Hereinafter this will be denoted by "virus having antigenic properties" or more briefly, "virus" or "virus antigen".
"Immunoglobulins" will refer to protein molecules from an animal species and having either a simple antigen activity or a simple antibody activity or both activities simultaneously, i.e. antigenic and antibody. In the case of each animal species, both man and other animals (such as the goat, guinea-pig or rabbit), immunoglobulins belong to various immunochemical classes, i.e. immunoglobulins in a class called A--called IgA for short--, immunoglobulins in a class G (called IgG), etc. In general, an immunoglobulin in the immunochemical class X and from the animal species I will be denoted IgX I.
The term "test serum" used here denotes a serum (or solution) containing immunoglobulins from a certain species of animals and in a certain immunochemical class, e.g. class G human immunoglobulins (IgG) having a specific antibody activity towards a particular erythrocyte or cell antigen in a group of individuals from the same species, i.e. a group of human beings in the present example. We prefer here to speak of a group of individuals since, as any serologist knows, a single erythrocyte or cell antigen is not necessarily present in all individuals of a given species of animals but is usually present in only some individuals, which form a group.
Similarly the terms "test corpuscles", "test cells" or "test particles" refer to corpuscles, cells or particles having antigen units from a certain species of animals which may become fixed to immunogloubulins carrying the antibody unit corresponding specifically to the antigen units.
"Antiglobulin" denotes an immune serum from an animal species II different from a species I and supplying immunoglobulins having a specific antibody activity towards IgX I. The antiglobulin will be called Ig II anti-IgX I, or simply anti-IgX I if no confusion is possible.
2. The Prior Art
It is known to use "Coombs" reactions for determining the presence of antibodies (or erythrocyte antigens) in the plasma or serum of blood (or in erythrocytes). The known method is as follows, e.g. when determining whether the plasma of a sample of human blood contains a particular antibody, e.g. a specific anti-D antibody, or in other words contains immunoglobulins specific to the "D" antigen, some of which are in the immunochemical class G and will therefore be called "IgG". The plasma to be analysed is incubated in a tube with erythrocytes carrying D antigen units, i.e. erythrocytes having an antigen activity specific to the antibody for which a search is being made. Any immunoglobulins of the specific antibody present in the plasma will become fixed, during the contact period, on to the corresponding antigen units on the corpuscles. The resulting reaction medium is washed and then incubated with an antiglobulin, e.g. a goat serum providing anti-human IgG immunoglobulins. During the incubation, a goat immunoglobulin supplied by the antiglobulin, owing to its specific anti-human unit becomes fixed to any human immunoglobulin which has become fixed to an antigen unit specific to an erythrocyte. After the incubation, the reaction medium is centrifuged; if the specific anti-D antibody searched for was actually present in the plasma, this results in agglutination of the erythrocytes produced by the immunoglobulins provided by the antiglobulin, as a result of the production of the following chains of bonds:
Erythrocyte (D antigen)/specific anti-D human immunoglobulin/anti human-immunoglobulins/specific anti-D human immunoglobulin/(D antigen) erythrocyte. The agglutination occurs in the form of a deposit of agglutinated erythrocytes at the bottom of the tube, but the deposit is not always easy to distinguish from a deposit of non-agglutinated erythrocytes. The two deposits are distinguished by gently agitating the tube contents. In negative reactions, i.e. reactions brought about on the plasma of blood not containing the specific anti-D antibody, the non-agglutinated erythrocytes return to homogeneous suspension, whereas in positive reactions the erythrocytes continue to stick together in relatively large groups.
This method involves relatively long, complicated reactions, and sometimes these reactions lack sensitivity, which may be dangerous.
The problem of exactly determining the nature of the antigens carried by a cell also occurs when transplanting organs. It is known that lymphocytes carry a certain number of antigens called "HLA"-system antigens. Before any surgical transplantation, it is essential to determine which HLA antigens are carried by the donor's and recipient's lymphocytes, in order to ensure that there is at least theoretical histo-compatibility between the lymphocytes from these two different individuals, since otherwise it will be impossible to transplant an organ. Owing to the large number of different HLA-system antigens which may be carried by lymphocytes, a large number of elementary reactions (e.g. 100) have to be carried out to determine the specific nature of the HLA antigens present. These operations are all the more difficult in that lymphocyte cells are available only in very small quantities and can thus be used only in microreactions.
Nowadays some viruses or virus antigens (e.g. HB.sub.s antigen) are detected by radio-immunological methods which are very expensive and cause pollution and therefore require special authorization.